Breast cancer is one of the most common malignancies in women, and is the leading cause of death among women between the ages of 40 and 55 years in the United States. During the last two decades, this cancer has been studied intensively, and recently new preventive measures and therapies have emerged, especially immunological and genetic treatments administered as adjuvant therapy after surgery, radiation, and chemotherapy. Biotherapies have produced successful results in mice with mammary carcinoma, particularly with cellular vaccines, DNA vaccines, recombinant proteins, and adoptive immunotherapy.
The progression of breast cancer is often accompanied by changes in gene expression patterns in cells of growing carcinomas, resulting in highly tumorigenic and invasive cell types. Thus, AP-1 transcription factor (Activating Protein-1) belongs to a group of factors, which define tumor progression and regulate breast cancer cell invasion and growth, as well as resistance to anti-estrogens. In addition, Fra-1 (Fos-related antigen-1), a transcription factor belonging to the AP-1 family, is overexpressed in many human and mouse carcinoma cells, including those of thyroid, kidney, esophagus and breast. Overexpression of Fra-1 in epithelial carcinoma cells greatly influences their morphology, motility and invasiveness, and activates the transcription of a number of genes. Overexpression of this transcription factor also correlates with transformation of epithelial tumor cells to a more invasive phenotype, and a close, specific association of Fra-1 expression with highly invasive breast cancer cells has been reported. Taken together, these findings suggest that overexpressed Fra-1 can serve as a potential target for active vaccination against breast cancer.
Interleukin-18 (IL-18) is a potent immunoregulatory cytokine that was initially described as an IFN-γ inducing factor. This cytokine also enhances cytokine production of T cells and/or natural killer (NK) cells and induces their proliferation and cytolytic activity. Tumor cells engineered to produce IL-18 are less tumorigenic and systemic administration of IL-18 reportedly afforded considerable therapeutic activity in several murine tumor models. In addition, IL-18 enhances cellular immune mechanisms by upregulating major histocompatibility complex (MHC) expression and by favoring the differentiation of CD4+ helper T cells towards the Th1 subtype. In turn, Th1 cells secrete IL-2 and IFN-γ, which facilitate the proliferation and/or activation of CD8+ cytotoxic T lymphocytes, NK cells and macrophages, all of which can contribute to tumor regression. In addition, IL-18 is a novel inhibitor of angiogenesis, sufficiently potent to suppress tumor growth by directly inhibiting fibroblast growth factor-2 (FGF-2)-induced endothelial cell proliferation. Recombinant IL-18 has been evaluated as a biological “adjuvant” in murine tumor models, and its systemic administration induced significant antitumor effects in several tumor models.
Asada et al. have reported significant antitumor effects utilizing an autologous tumor cell vaccine engineered to secrete interleukin-12 (IL-12) and IL-18 in a viral vector (Molec. Therapy 2002, 5(5): 609-616).
Vaccines have been utilized to provide a long term protection against a number of disease conditions by very limited administration of a prophylactic agent that stimulates an organism's immune system to destroy disease pathogens before they can proliferate and cause a pathological effect. Various approaches to vaccines and vaccinations are described in Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, Second Edition, ASM Press pp. 253-276 (1998).
Vaccination is a means of inducing the body's own immune system to seek out and destroy an infecting agent before it causes a pathological response. Typically, vaccines are either live, but attenuated, infectious agents (virus or bacteria), or a killed form of the agent. A vaccine consisting of a live bacteria or virus must be non-pathogenic. Typically, a bacterial or viral culture is attenuated (weakened) by physical or chemical treatment. Although the agent is nonvirulent, it can still elicit an immune response in a subject treated with the vaccine.
An immune response is elicited by antigens, which can be either specific macromolecules or an infectious agent. These antigens are generally either proteins, polysaccharides, lipids, or glycolipids, which are recognized as “foreign” by lymphocytes known as B cells and T cells. Exposure of both types of lymphocytes to an antigen elicits a rapid cell division and differentiation response, resulting in the formation of clones of the exposed lymphocytes. B cells produce plasma cells, which in turn, produce proteins called antibodies (Ab), which selectively bind to the antigens present on the infectious agent, thus neutralizing or inactivating the pathogen (humoral immunity). In some cases, B cell response requires the assistance of CD4 helper T cells.
The specialized T cell clone that forms in response to the antigen exposure is a cytotoxic T lymphocyte (CTL), which is capable of binding to and eliminating pathogens and tissues that present the antigen (cell-mediated or cellular immunity). In some cases, an antigen presenting cell (APC) such as a dendritic cell, will envelop a pathogen or other foreign cell by endocytosis. The APC then processes the antigens from the cells and presents these antigens in the form of a histocompatibility molecule:peptide complex to the T cell receptor (TCR) on CTLs, thus stimulating an immune response.
Humoral immunity characterized by the formation of specific antibodies is generally most effective against acute bacterial infections and repeat infections from viruses, whereas cell-mediated immunity is most effective against viral infection, chronic intracellular bacterial infection, and fungal infection. Cellular immunity is also known to protect against cancers and is responsible for rejection of organ transplants.
Antibodies to antigens from prior infections remain detectable in the blood for very long periods of time, thus affording a means of determining prior exposure to a pathogen. Upon re-exposure to the same pathogen, the immune system effectively prevents reinfection by eliminating the pathogenic agent before it can proliferate and produce a pathogenic response.
The same immune response that would be elicited by a pathogen can also sometimes be produced by a non-pathogenic agent that presents the same antigen as the pathogen. In this manner, the subject can be protected against subsequent exposure to the pathogen without having previously fought off an infection.
Not all infectious agents can be readily cultured and inactivated, as is required for vaccine formation, however. Modern recombinant DNA techniques have allowed the engineering of new vaccines to seek to overcome this limitation. Infectious agents can be created that lack the pathogenic genes, thus allowing a live, nonvirulent form of the organism to be used as a vaccine. It is also possible to engineer a relatively nonpathogenic organism such as E. coli to present the cell surface antigens of a pathogenic carrier. The immune system of a subject vaccinated with such a transformed carrier is “tricked” into forming antibodies to the pathogen. The antigenic proteins of a pathogenic agent can be engineered and expressed in a nonpathogenic species and the antigenic proteins can be isolated and purified to produce a “subunit vaccine.” Subunit vaccines have the advantage of being stable, safe, and chemically well defined; however, their production can be cost prohibitive.
A new approach to vaccines has emerged in recent years, broadly termed genetic immunization. In this approach, a gene encoding an antigen of a pathogenic agent is operably inserted into cells in the subject to be immunized. The treated cells are transformed and produce the antigenic proteins of the pathogen. These in vivo-produced antigens then trigger the desired immune response in the host. The genetic material utilized in such genetic vaccines can be either a DNA or RNA construct. Often the polynucleotide encoding the antigen is introduced in combination with other promoter polynucleotide sequences to enhance insertion, replication, or expression of the gene.
Polynucleotide vaccines (also referred to as DNA vaccines) encoding antigen genes can be introduced into the host cells of the subject by a variety of expression systems. These expression systems include prokaryotic, mammalian, and yeast expression systems. For example, one approach is to utilize a viral vector, such as vaccinia virus incorporating the new genetic material, to innoculate the host cells. Alternatively, the genetic material can be incorporated in a plasmid vector or can be delivered directly to the host cells as a “naked” polynucleotide, i.e. simply as purified DNA. In addition, the DNA can be stably transfected into attenuated bacteria such as Salmonella typhimurium. When a patient is orally vaccinated with the transformed Salmonella, the bacteria are transported to Peyer's patches in the gut (i.e., secondary lymphoid tissues), which then stimulate an immune response.
Polynucleotide vaccines provide an opportunity to immunize against disease states that are not caused by traditional pathogens, such as genetic diseases and cancer. Typically, in a genetic cancer vaccine, antigens to a specific type of tumor cell must be isolated and then introduced into the vaccine. An effective general vaccine against a number of cancers can thus entail development of numerous individual vaccines for each type of cancer cell to be immunized against. There is an ongoing need and desire, therefore, for vaccines that can stimulate a general immune response against a variety of cancer cells.
The present invention fulfills the ongoing need for vaccines that can stimulate a general immune response against cancer cells, such as breast cancer cells, by providing a DNA vaccine encoding a Fra-1 protein and IL-18 in a single host vector.